Scanning laser device and methods of use

ABSTRACT

In one aspect, the invention provides vision prosthesis systems. Exemplary vision prosthesis systems of the invention comprise a light energy generator operably connected to a wearable head piece comprising a device for directing light energy produced by the light energy generator onto a mammalian retina, wherein the light energy generator is tuned to emit light energy of sufficient power to modulate neural activity in the retina. In another aspect, the invention provides methods for irradiating neurons in the retina of the mammalian eye by directing light energy produced by a light energy generator onto a mammalian retina. The methods of the invention may be used to directly modulate the activity of retinal neurons or to introduce molecules into retinal cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/888,428, filed Jul. 9, 2004, which claims the benefit of U.S.Provisional Application No. 60/486,623, filed Jul. 11, 2003, under 35U.S.C. §119.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with government support under 9978888 awarded bythe National Science Foundation. The government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention relates to methods and prosthetic apparatus formodulating neural activity and in particular to apparatus for producingan artificially-formed sense, such as vision.

BACKGROUND

The mammalian eye includes a cornea, a fluid-filled anterior chamber, alens, a fluid-filled posterior chamber, and a photosensitive retina. Thedesignations “anterior” and “posterior” are with reference to the lens.Light enters the eye through the cornea, passes through the anteriorchamber and is focused onto the retina by the lens. The retina iscomposed of numerous types of cells including photosensitive rods, thatare responsible for night vision, and photosensitive cones that areresponsible for high acuity day vision. The rods and cones communicatewith neurons in the retina that transmit nerve impulses to the visualcenters of the brain where this information is processed to generate theperception of visual information. For example, rods and conescommunicate with bipolar cells, that, in turn, communicate with ganglioncells that transmit nerve impulses to the brain.

Half of all blindness is caused by damage or disease of the retina(Zrenner (2002) Science 295(5557):1022-26). Death of photoreceptors hasmany causes, some of which are the results of known, underlying, geneticmutations, while others are of unknown cause. In the condition known asretinitis pigmentosa, the rods degenerate first and result in loss ofnight vision, and later the cones degenerate, resulting in totalblindness. In the condition known as age-related macular degeneration,the cones degenerate, and since they are necessary for high acuityvision, these individuals also become functionally blind. In bothdiseases the non-photoreceptive neurons, such as the bipolar andganglion cells, remain largely intact (Santos et al. (1997) Arch.Ophthamol. 115:511-5; Kim et al. (2002) Retina 22:464-70; Kim et al.(2002) Retina 22:471-7). Due to the many applications of lasers and theincreasing use in the military, laser eye injuries that destroy theretinal pigmented epithelium and light-sensitive photoreceptors are agrowing problem (Barkana & Belkin (2000) Surv. Opthalmol.44(6):459-478). The retinal pigmented epithelium is located between thephotoreceptors and the choroid of the eye. Certain laser eye injuriespreserve the functionality of the retinal neurons, so that the use ofvision prostheses become a viable option (e.g., krypton laser damage,Weiland et al. (1999) Graefe's Arch. Clin. Exp. Opthalmol. 237:1007-13).

There are currently no treatments for most eye injuries and diseasesthat cause the destruction and degeneration of photoreceptors. Therehave been attempts, however, to develop a retinal prosthesis by theimplantation of multiple active electrodes which sense light impingingon the retina and generate corresponding electrical signals thatstimulate the healthy retinal neurons in the eye (see, e.g., Dagnelie &Massof (1996) IEEE Spectrum, May 22-29; Chase (1999) Technol. Rev.102:44-48; Haystead (1999) Vision Systems Design 4(6):31-36; Chow et al.(2002) J. Rehab. Res. Dev. 39(3):313-22; Humayun et al. (2003) VisionRes. 43(24):2573-81). Implanted electrode arrays have significantlimitations, however, such as power dissipation, incompatibility withsurrounding biological tissues, low density of photocells in theelectrode arrays, and the lack of knowledge about the neuralinterconnections to the bipolar and ganglion cells, and the type ofstimulation required to mimic retinal signal processing (Liu et al.(2000) IEEE J. Solid-State Circuits, 35(10):1487-1497; Zrenner (2002)Science 295(5557):1022-26).

Thus, there is a continuing need for less invasive prosthetic devicescapable of stimulating, inhibiting, or otherwise modulating, theactivity of retinal neurons, in damaged and diseased retinas in whichthe photosensitive cells are functionally impaired, in order to generatea pattern of nerve impulses that are transmitted to the visual centersof the brain to yield visual information. There is also a need formanipulating the mammalian retina to promote health, for example, bystimulating retinal cells to take up molecules such as nucleic acids,proteins, and nutrients.

SUMMARY

In one aspect, the invention provides vision prosthesis systems.Exemplary vision prosthesis systems of the invention comprise a lightenergy generator operably connected to a wearable head piece comprisinga device for directing light energy produced by the light energygenerator onto a mammalian retina, wherein the light energy generator istuned to emit light energy of sufficient power to modulate neuralactivity in the retina. The light energy generator may be tuned to emitinfrared, visible, or ultraviolet light energy. For light energy betweenabout 400 and 700 nm wavelengths, the power of the light energy isgenerally greater than about 1 mW. In some embodiments, the light energygenerator is tuned to emit pulses of light energy. The light energygenerator may be selected from the group consisting of a laser, a laserdiode, a mercury arc lamp, and a light-emitting diode. In someembodiments, the light energy generator is a femtosecond-pulsed infraredlaser or a picosecond-pulsed infrared laser.

The device for directing light energy onto the mammalian retina may be ascanner. Suitable scanners for use in the systems of the inventioninclude, but are not limited to, fiber optic scanners, mirror scanners,and acousto-optical deflectors. In some embodiments, the device fordirecting light energy onto the retina is a non-resonant scanner.

The systems of the invention may further comprise an image source, forexample, a camera, operably connected to the image processor. In someembodiments, the systems of the invention are controlled by a computer.

In some embodiments, the invention provides a vision prosthesis system,comprising:

a light energy generator tuned to emit light energy of sufficient powerto modulate neural activity in the retina, wherein the power level oflight energy emitted between about 400 and 700 nm wavelengths is greaterthan about 1 mW;

a wearable head piece comprising a scanner and an ocular lens, whereinthe scanner is operably connected to the light energy generator andconfigured for scanning light energy produced by the light energygenerator onto a mammalian retina, and wherein the ocular lens isoperably connected to the scanner for focusing the light energy onto theretina; and

an image processor operably connected to an image source and configuredto transmit electrical signals corresponding to an image to the scanner.

A further embodiment of a visual prosthesis system of the inventioncomprises a wearable head piece comprising a non-resonant scanneroperably connected to a light energy generator, wherein the light energygenerator is tuned to emit light energy of sufficient power to directlymodulate the activity of neurons in a mammalian retina and wherein thescanner is configured for scanning the light energy onto the mammalianretina.

Another aspect of the invention provides methods for irradiating neuronsin the retina of the mammalian eye. In some embodiments, the methods ofthe invention comprise the step of directing light energy onto amammalian retina using a visual prosthesis system to directly modulatethe activity of a retinal neuron, the visual prosthesis systemcomprising a light energy generator operably connected to a wearablehead piece comprising a device for directing light energy produced bythe light energy generator onto a mammalian retina, wherein the lightenergy generator is tuned to emit light energy of sufficient power tomodulate neural activity in the retina. Some embodiments provide methodsmodulating neural activity in the retina of a mammalian eye, comprisingthe step of directing light energy produced by a light energy generatoronto a mammalian retina to directly modulate the activity of retinalneurons. Further embodiments provide methods for introducing a moleculeinto a retinal cell in vivo, comprising the step of directing lightenergy produced by a light energy generator onto a mammalian retina invivo, wherein the retinal cell comprises a plasma membrane and the lightenergy has a power sufficient to permeabilize the plasma membrane,thereby allowing a molecule outside the retinal cell to enter theretinal cell. The methods for introducing a molecule into a retinal cellin vivo may comprise the steps of (a) contacting a cell having a plasmamembrane in a mammalian retina with a molecule in vivo; and (b)directing light energy produced by a light energy generator onto themammalian retina, wherein the light energy has sufficient power topermeabilize the membrane of the contacted cell in the mammalian retina,thereby allowing the molecule to enter the cell. Exemplary molecules foroptoporating into retinal cells include, but are not limited to,nutrient molecules, proteins, nucleic acids, and lipids.

The light energy used in the methods of the invention may be produced bya light energy generator selected from the group consisting of a laser,a laser diode, a mercury arc lamp, and a light-emitting diode. Forexample, the light energy may be produced by a femtosecond-pulsedinfrared laser or a picosecond-pulsed infrared laser.

In some embodiments, the light energy is directed onto the retina usinga scanner. For example, light energy may be scanned onto the retina inraster format or in vector graphics format. In some embodiments of themethods of the invention the eye comprises an artificial lens to focusthe light energy onto the retina.

The systems and methods of the invention are useful, for example formodulating the activity of neurons in damaged or diseased retinas inwhich the photosensitive cells are functionally impaired in order togenerate a visual image. Another exemplary use for the methods of theinvention is for introducing molecules into retinal cells in order topromote the health and survival of the retina.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 depicts one exemplary embodiment of a system for practicingfemtosecond-pulsed to picosecond-pulsed near-infrared laser excitationaccording to the present invention;

FIG. 2 depicts an alternative embodiment of the invention for visiblelaser diode single-photon excitation;

FIG. 3 illustrates another embodiment of a visual prosthesis of theinvention that uses high-power UV or visible light emitting diodes(LEDs) to produce the stimulating light energy;

FIG. 4 depicts a means to increase the laser energy per image projectedonto the retina by conversion of the scanned image from the standardraster to a vector graphics format. The scan distance is significantlyreduced using the vector graphics format (about 90% in the exampleshown), allowing more light exposure per pixel assuming constant opticalpower of the illumination beam;

FIG. 5 depicts an alternative means of increasing laser energy densityto the retina by having an intraocular lens (IOL) to more efficientlyfocus the laser energy at the retinal neurons;

FIG. 6 depicts an array of micro light emitting diodes (micro-LEDs) ormicro-lasers that would be head-mounted and the image of the lightsources be projected onto the retina. Optical rays from three sourcesare shown. The array of light sources may be replaced with a spatiallight modulator.

DETAILED DESCRIPTION

In one aspect, the present invention provides a vision prosthetic systemthat directly modulates neural activity in the retina of a mammalianeye, such as a human eye. The prosthetic system is configured toirradiate the retina of a mammalian eye with light energy. In someembodiments, the vision prosthesis system comprises a light energygenerator operably connected to a wearable head piece comprising adevice for directing light energy produced by the light energy generatoronto a mammalian retina, wherein the light energy generator is tuned toemit light energy of sufficient power to modulate neural activity in theretina.

As used herein, the term “light energy” includes infrared (IR), visibleand ultraviolet (UV) light energy. Thus, the wavelengths of the lightenergy may be the in the range of about 300 nm to about 1800 nm.Exemplary power levels applicable to light energy of wavelengths betweenabout 300 nm and about 1800 nm are between about 2 mW and about 500 mW.For light energy with a wavelength between about 400 and 700 nm, thepower level entering the eye is generally greater than about 1 mW. Apower level in the range of 1 mW is much higher than the maximumpermissible exposure limits for long-term viewing of a stationary laserbeam within the visible spectrum. Typically, individual cells within theretina are exposed to less than 1 second duration of continuous lightexposure. In some embodiment, the light energy is pulsed. Pulsing thelight energy may increase the peak power of the light energy generatorand may also reduce heating of the retina. For example, the light energymay be pulsed while the beam entering the eye is not scanning. In otherembodiments the pulsed or continuous-wave light energy is scanned acrossthe retina, thereby limiting the exposure time of individual cellswithin the retina.

The light energy is directed onto the neuronal layer of a mammalianretina by a device suitable for the delivery of the light energy to theretina (e.g., a scanner) and the focusing ability of the natural opticsof the eye and, optionally, corrective optics. At the focal point of thelight energy beam, the retinal neuron responds to the focusedillumination resulting in a modulation of neural activity. The term“modulating neural activity in the retina” encompasses any change in thefrequency, intensity, and/or pattern of nerve impulses in any type ofneuron (e.g., bipolar cells and ganglion cells) in the retina. Forexample, the frequency of nerve impulses in one or more types of neuronsin the retina of a mammalian eye can be increased or decreased bytreating the retinal neurons in accordance with the methods of thepresent invention. According to the invention, the modulation of neuralactivity in the retina can occur directly, that is, in the absence ofphotochemical signal transduction mediated by photoreceptors.

In another aspect, the invention provides methods for irradiatingneurons in the retina of a mammalian eye, comprising the step ofdirecting light energy onto a mammalian retina using a visual prosthesissystem, the visual prosthesis system comprising a light energy generatoroperably connected to a wearable head piece comprising a device fordirecting light energy produced by the light energy generator onto amammalian retina. In some embodiments, the light energy generator istuned to emit short pulses of light energy of wavelengths between about700 nm and about 1100 nm (the NIR ‘optical window’ range whereabsorption within human tissue is a minimum). In some embodiments, thepower level of the light energy emitted by the NIR light energygenerator is between about 2 mW and about 500 mW.

Some embodiments of the invention provide methods for modulating neuralactivity in the retina of a mammal, wherein the methods each comprisethe step of directing light energy produced by a light energy generator(e.g., laser light energy) onto a mammalian retina to directly modulateneural activity in the retina. Thus, the light energy generator is tunedto emit light energy of sufficient power to modulate neural activity inthe retina. The modulation of neural activity in the retina may generatethe sensation of vision in the mammal. The irradiated retina does notinclude exogenous (i.e., introduced into the retina by artificial means)photoactivatable precursor molecules that are activated by light torelease a neuroactive molecule that interacts with a neural cell in theretina, thereby modulating activity of the neural cell. The methods ofthe present invention rely, instead, on the direct interaction of lightwith neural cells in the retina in the absence of photochemical signaltransduction mediated by photoreceptors. While not wishing to be boundby theory, it is hypothesized that sufficiently energetic light wavescauses the temporary formation of pores in the cell membrane of neuralcells, thereby resulting in membrane depolarization and the generationof action potentials that transmit nerve signals to the visual cortex ofthe mammalian brain thereby generating a visual image. An alternativemechanism for the modulation of neural activity may be that lightchanges the electric field across the cell membrane, therebydepolarizing or hyperpolarizing the cell (Hirase et al. (2002) J.Neurobiol. 51:237-47).

In further embodiments, the invention provides methods for introducingmolecules into retinal cells in vivo by optoporation. In someembodiments, the methods comprise the step of directing light energyproduced by a light energy generator onto a mammalian retina in vivo,wherein the retinal cell comprises a plasma membrane and the lightenergy has an intensity sufficient to permeabilize the membrane of aretinal cell, thereby allowing a molecule outside the retinal cell toenter the retinal cell. The molecule outside the retinal cell may be anendogenous molecule, such as a nutrient molecule. In some embodiments,the methods comprise the steps of: (a) contacting a cell in a mammalianretina with a molecule in vivo; and (b) directing light energy producedby a pulsed light energy generator onto the mammalian retina, whereinthe light energy has an intensity sufficient to temporarily permeabilizethe membrane of the contacted cell in the mammalian retina, therebyallowing the molecule to enter the cell. Exemplary molecules useful foroptoporating into retinal cells include nutrient molecules, proteins,nucleic acids, lipids, etc. For example, the methods may be used tooptoporate nucleic acid molecules encoding a protein, as described inEXAMPLE 1. Other suitable nucleic acids for optoporation into retinalcells include, but are not limited to, antisense RNA, small-interferingRNA, and modified nucleic acids.

In some embodiments, the optoporation methods of the invention are usedto introduce molecules that promote the survival of the retinal cellsand prevent further degeneration in the diseased retina. Exemplarymolecules that are suitable for promoting survival and preventingdegeneration of the retina include, but are not limited to, growthfactors or nucleic acid molecules coding for growth factors, such asneurotrophins (e.g., Nerve Growth Factor, NGF, Brain DerivedNeurotrophic Factor, BDNF, Neurotrophin-3, NT-3) and small moleculesthat act directly on second messenger signal transduction cascadesdownstream of the growth factors, such as TPA to activate the proteinkinase C signaling cascade. The optoporation methods of the inventionmay also be used to introduce molecules that act on the ion channels ofthe cell from the intracellular side of the plasma membrane, tostimulate the cells to fire action potentials, either through theirdirect action on the channel, or through the activation of a secondmessenger cascade, such as cyclic-AMP or cyclic-GMP, which then acts tomodulate channel conductance.

Prior to discussing various exemplary embodiments of a prosthetic deviceconstructed according to the invention, it is appropriate to firstdiscuss various biological components of an eye that interact with thelight energy delivered by the prosthetic device. Neurons that arelocated in the retina of a mammalian eye include, but are not limitedto, bipolar cells (including ON and OFF subtypes, and rod and conesubtypes), ganglion cells (including ON and OFF subtypes, bistratifiedsubtype, red/green color opponent subtype, and blue/yellow coloropponent subtype), horizontal cells and amacrine cells (which includenumerous subtypes).

According to the methods of the invention, retinal neural activity ismodulated both nondestructively and with minimal-invasiveness byirradiating the retinal neuronal layer with light energy, for examplepulses of light energy. Irradiation of the retinal neuronal layers canbe achieved by any useful light energy generator. Exemplary light energygenerator that are useful in the practice of the present inventioninclude, but are not limited to, lasers, laser diodes, mercury arclamps, and light-emitting diodes (LEDs). In some embodiments, the lightenergy generator is a laser, as described in more detail below. Laserscan be tuned to a desired wavelength of light, and can deliver light tothe retina at very high resolution and intensity from a single opticalfiber. Typically, a single laser is tuned to deliver light at anelectro-magnetic frequency, duration, power, and repetition rate that isoptimal for modulating neural activity. However, two or more laserstuned at different wavelengths may also be used. Single photon ormulti-photon (e.g., two or three photon) excitation may be used (He etal. (2002) Nature 415:767-70). Effective three-photon suppression toavoid unwanted high-energy absorption has been demonstrated byphase-controlled multi-photon interference (Lozovoy et al. (2003) J.Chem. Phys. 118(7):3187-96), which may be used to selectively excite thes, m, and 1 cones (color vision). Exemplary ranges for single-photonultraviolet (UV)-violet excitation include wavelengths between about 300nm to about 450 nm, such as about 315 nm to about 400 nm or about 325 nmto about 425 nm. Exemplary ranges for multi-photon deep-red to infraredexcitation include wavelengths between about 650 nm to about 1800 nm,such as about 650 nm to about 1200 nm wavelengths for two-photonphotostimulation or about 900 nm to about 1800 nm for three-photonexcitations.

In some embodiments of the present invention, electrical signals (thatencode information that represents a visual image) are processed todirect a scanner to scan a beam of light produced by a light source ontothe retina, thereby forming a pattern of light intensities on theretina. Exemplary scanners that are suitable for use according to theinvention include, but are not limited to, fiber optic scanners, mirrorscanners, and acousto-optical deflectors. The scanners may be resonantscanners (e.g., raster scanning) or non-resonant scanners (e.g., vectorgraphics scanning). Most optical scanners for low-power applicationssuch as visual displays use raster scanning, where the entire field isscanned and both on and off pixels being scanned. However, forapplications that require higher optical power per on-pixel, vectorgraphics scanning or pixel-addressable scanning is desirable. Typically,a resonant scanner cannot be used effectively for a vector graphicsscanning, since the scanner mechanism is designed to run at a fixedfrequency and a constant scan amplitude, while vector graphics scannerstypically operate at various rates and amplitudes.

The light energy is scanned onto the retina to directly modulate theactivity of the retinal neurons. The amount of neural activity isproportional to the dose of irradiation delivered. Thus, the pattern oflight intensities generates a corresponding pattern of nerve impulseswithin the retina that are communicated to the brain, which uses thisinformation to construct a mental picture of the visual image (i.e., thebrain “sees” the visual image). In some embodiments, the light sourcegenerates a beam of light of sufficiently high resolution to generate avisual image that approximates, or equals, the resolution of normalsight.

Thus, in some embodiments, the present invention provides a system formodulating neural activity, particularly in the retina of a mammalianeye, wherein light energy produced by a light energy generator isdirected onto the retina in a pattern corresponding to the image to beperceived. In embodiments where a scanner is used to direct the lightenergy onto the retina, the scanning pattern may be a standard repeatingraster pattern or in a scan pattern that follows the outline of theobjects to be conveyed to the brain (e.g., vector scanning graphicalformat).

FIG. 1 illustrates one exemplary embodiment of a system 10 constructedaccording to the present invention. System 10 includes a wearableheadpiece 12 with components, discussed below, capable of directinglight energy to the user's retina. Briefly summarized, light energy isemitted from a pulsed light energy generator 14 that passes the lightenergy through an electronic shutter and modulator 16 to a groupvelocity dispersion compensator 18. The light energy is focused by alens 20 and thereafter conducted through a fiber optic cable 22. In theembodiment shown in FIG. 1, fiber optic cable 22 is attached to acabling disconnect 24, which releasably connects cable 22 to fiber opticcable 26. Fiber optic cable 26 terminates in an optical fiber scanner28. Light energy emerging from optical fiber scanner 28 is directed viaa mirror or beam splitter 30 to an ocular lens 32 that focuses the lightenergy onto the retina 34 of the user of headpiece 12.

Light energy generator 14 generally includes lasers that providesub-picosecond pulses of near-infrared (NIR) light that repeat atgreater than 50 MHz. Such femtosecond-pulsed lasers have been used foroptoporation and more commonly for multi-photon (e.g., two-photon)excitation of dyes and fluorophores. The most common form of thesefemtosecond-pulsed lasers are the titanium-sapphire laser manufacturedby Spectra Physics Inc. (Mountain View, Calif.) and Coherent Inc. (SantaClara, Calif.). In one example, the lasers may be comprised of thesecurrently available femtosecond-pulsed lasers that produce light energyin the near infrared (NIR) optical spectrum (690-1050 nm). A suitablepulsed laser in one embodiment of the invention may produce a pulsetrain of 100 femtosecond pulses at a repetition rate of 80 MHz at 790nm. Light energy generator 14 may include more than one operably linkedlasers; for example, a green pump laser may be operably linked to aTi:Sapphire laser to produce the light energy output. Short NIR pulsesof higher peak powers but at lower pulse repetition rates (<1 MHz) canbe generated by adding a cavity dumping oscillator to the Ti:Sapphirelaser output or having a Ti:Sapphire regenerative amplifier, such asRegA 9000/9050 by Coherent, Santa Clara, Calif. (see Theer et al. (2003)Optics Lett. 28(12):1022-4).

Electronic shutter and modulator 16 is controlled by electrical signalsfrom an image processor 40 and serves to modulate the intensity of thebeam of light energy communicated to the wearer's retina. Electronicshutter and modulator 16 may also interrupt the light energy produced bypulsed light energy generator 14, thereby producing light and dark areason the user's retina when the light energy is scanned onto the retina.The electronic shutter may also be used without a modulator, making abinary image without grayscale for the user of the prosthetic device. Inone embodiment, electronic shutter and modulator 16 is suitablycomprised of an acousto-optical modulator or Pockels Cell.

Group velocity dispersion compensator 18 preconditions the light pulsesto compensate for known degradation of the light pulses that occurs asthe light pulses are communicated to headpiece 12 and beyond to theretina. Group velocity dispersion compensator 18 may be suitablycomprised of optical prisms or diffraction gratings (Fan et al. (1999)Biophysical J. 76:2412-2420; Helmchen et al. (2001) Neuron 31:903-912).The preconditioned light pulses emerging from group velocity dispersioncompensator 18 are directed via lens 20 to optical fiber 22. Opticalfiber 22, in one embodiment, is a singlemode optical fiber.Microstructured optical fibers (photonic crystal fibers) have beendeveloped that allow higher power transmission of IR femtosecond-pulsedlaser light, eliminating the need for the group velocity dispersioncompensation for preservation of femtosecond pulse quality over metersof fiber length (Ouzounov et al. (2002) CLEO 2002, 455; Myaing et al.(2003) Optics Lett. 28(14):1224-6). These optical fibers arecommercially available from Crystal Fibre (Lyngby, Denmark), BlazePhotonics (Bath, England), and OmniGuide Communications Inc. (Cambridge,Mass.).

FIG. 1 depicts an optical fiber scanner 28 that provides two dimensionalX-Y scanning of the light energy emerging from headpiece optical fiber26, thereby producing a two dimensional image on the user's retina.Optical fiber scanner 28 may employ piezoelectric elements that moveaccording to arbitrary voltages applied to the X and Y actuatorscomputer-controlled vector scanning, or move according to fixed voltageand frequency that create resonant X & Y scanner motions for fixedraster scanning. Alternative systems of the invention may suitably usemirror scanning to produce two-dimensional images on the user's retina.In mirror scanning, piezoelectric, electro-magnetic, or electrostaticelements may be used to control the position of a mirror that directsthe light energy in both X and Y directions to produce a scanned image.In both optical fiber scanning and mirror scanning, the light energy isused according to the invention to directly modulate retinal neuralactivity according to the pattern of light across two-dimensions. Thespatial pattern of modulated nerve firings will communicate the “image”to the user's brain.

Image processor 40 generates electrical signals that control bothelectronic shutter and modulator 16 and a scan controller 48. Imageprocessor 40 may be comprised of one or more computer processingelements that internally generate an image signal or receive an imagesignal from an external source 41, such as a television, computer,digital video disk (DVD) player, etc. In one embodiment, an optionalbody-mounted camera 42 produces either still or continuous video imagerythat is communicated to image processor 40 via wires 44, 46, and scancontroller 48. Image processor 40 causes electronic shutter andmodulator 16 to output light energy pulses that are scanned onto theuser's retina. Image processor 40 controls the scanning of the lightpulses by way of drive signals from scan controller 48. Another functionof image processor 40 and scan controller 48 is to re-map the pixelsfrom the source, such as a video camera 42 or alternative raster-scannedimage source 41 (e.g., television, computer, or DVD player), into avector graphics format so the delivered neural stimulation is mosteffective (see FIG. 4).

Scan controller 48 communicates electrical signals to optical fiberscanner 28 via wire 46, which connects through cabling disconnect 24 towire 38. Wire 38 is, in turn, connected to optical fiber scanner 28.Sensors used to detect scan position of moving optical fiber scanner 28can be used to reduce scanner-induced distortion in the image. Sensorsused to detect when the eye is looking straight ahead can be used totrigger the shutter so the laser excitation is maintained on the sameregion of the retina.

Wearable headpiece 12 may further include a spectacle lens 36. Lens 36may suitably provide a cosmetic effect for the user by shielding thecomponents of the system 10 that are disposed behind lens 36. Lens 36may also be darkened or made reflective to produce a dark environmentfor the user's eyes. An optional head strap may be used to attachheadpiece 12 to the user's head.

Examples of lasers used for pulsed light energy generator 14 may includethe MaiTai or Millennium and Tsunami models of Ti-Sapphire lasersmanufactured by Spectra Physics of Mountain View, Calif. or the Mira andChameleon models manufactured by Coherent of Santa Clara, Calif.Recently, diode-based, femtosecond-pulsed NIR laser systems have becomeavailable that are much smaller in size and lower in cost compared tothe standard Ti-sapphire laser systems identified above (Hogan,Biophotonics Intl. May 2003, p. 42-7). Diode-based, single IR wavelengthfemtosecond-pulsed lasers have been engineered to be battery-powered,small, and portable (Agate et al. (2002) Optics Comm. 205:207-13).Therefore, the embodiment that uses femtosecond-pulsed NIR lasers may beuseful in embodiments of the present invention that are completelywearable and portable.

Lasers currently contemplated for the embodiment shown in FIG. 1 producelight energy in pulsed beams that may be transmitted at lower powerthrough the eye. The photons in each of the light beams are directed orfocused in system 10 to produce a high-intensity spot, on the user'sretina, of high enough power density to directly activate the neurons.An advantage of this embodiment of the invention is that each of thelight beams does not have sufficient power density or intensity tophotostimulate receptors in the user's eye until the beams are focusedon a particular spot. This means that surrounding ocular tissue andneurons in the user's eye are not activated by the beams, therebyproducing higher contrast in the stimulated image than usingphotostimulation based on linear, single-photon processes. The majorabsorber of light in the diseased eye is the retinal pigmentedepithelium that contains melanin. Melanin has maximal absorbance at 500nm and significantly less, possibly negligible, absorption in the nearinfra-red (>700 nm) for single-photon absorption, see Roderick (1973)The Vertebrate Retina, page 182).

FIG. 2 illustrates another embodiment of system 10′ having similarelements identified by the same reference numerals in FIG. 1. Theprimary difference in the embodiment shown in FIG. 2 is pulsed lightenergy generator 14′. In FIG. 2, pulsed light energy generator 14′ iscomprised of one or more laser diodes that each produce light energy inthe near UV to visible wavelengths. UV and violet (350-420 nm) laserdiodes recently introduced by Nichia Corporation of Tokushima, Japan maybe useful in embodiments of the present invention that are completelywearable and portable. Laser diodes producing light energy of greaterthan 20 mW optical power at 405±10 nm were introduced by NichiaCorporation in 2000 and laser diodes producing light energy at 370-380nm were introduced in 2002. UV and violet laser diodes have enoughenergy per photon to directly stimulate the retinal neurons. Longerwavelength green and red solid state and laser diodes are commonplaceand may provide the required laser power for the direct modulation ofretinal neurons.

Notably, system 10′ illustrated in FIG. 2 does not include electronicshutter and modulator 16 and group velocity dispersion compensator 18.This is because the laser diodes in pulsed light energy generator 14′are directly modulated by image processor 40. The laser diodes may bedirectly connected (i.e., “pigtailed”) to optical fibers 50, 52 thatcommunicate the light energy to the left and right eyes, respectively.Exemplary devices for pigtailing to optical fiber are available fromMicro Laser Systems of Garden Grove, Calif. The optical fibers 50, 52are connected to optical fibers 54, 56 via the cabling disconnect 24.For purposes of redundancy or safety, multiple laser diodes of lowerintensity may be coupled to optical fibers 50, 52. The output of the twoor more laser diodes can be combined by either polarization or dichroicbeam splitters before coupling to the optical fiber. Since the lasermodulation produces pulses of relatively long duration for single-photonstimulation versus multiphoton excitation, compensation for groupvelocity dispersion is not needed.

Camera 42 in FIG. 2 is also shown with an alternative direct wiring 58to image processor 40, instead of using the path comprised of wires 44,46, and scan controller 48. Wiring 58, and other wiring discussedherein, may also be accomplished using wireless signal communication,where it is technologically feasible and desired.

Systems 10 and 10′ in FIGS. 1 and 2 depicts a wearable neuro-stimulatingprosthesis providing single or multi-photon laser excitation usingeither diode-based or femtosecond-pulsed lasers. Both single andmulti-photon lasers discussed herein can use the same optical fiber,such as a singlemode optical fiber for the near-infrared wavelengths.They can also use the same mirrors and fiber scanners, and the samedelivery optics for directing the light energy to the retinal neurons.

Cabling disconnect 24 depicted in FIGS. 1 and 2 permits a user to switchfrom a larger femtosecond-pulsed NIR laser system at home or at work toa portable, single-photon laser system (and visa versa). In that regard,cabling disconnect 24 is configured to provide the same interface to theoptical fibers extending from the laser sources of both systemconfigurations.

FIG. 3 illustrates another embodiment of a visual prosthesis of theinvention that uses high-power UV or visible light emitting diodes(LEDs) to produce the stimulating light energy. In FIG. 3, system 60 isshown from a top view, including a top view of the head of the wearer ofthe headpiece. Attached to the headpiece are cameras 62 connected to animage processor 64. Electrical signals from image processor 64 areamplified and modified by a scan controller 66. Image processor 64 alsocontrols a light energy generator 68, which in this embodiment iscomprised of high-power visible LEDs. The light energy produced by thelight energy generator 68 is communicated via optical fibers 70 totwo-dimensional X-Y minor scan systems 72. The X-Y mirror scan systemsinclude piezoelectric elements that cause mirrors in mirror scan systems72 to direct the light energy to form a scanned pattern of lightintensities on the user's retina. Alternative actuation methods areemployed for generating X-Y minor scanning in resonance andnon-resonance motion by manufacturers of retinal scanning displays(Microvision Inc., Bothell, Wash.) and optical switching devices (MEMSOptical Inc., Huntsville, Ala.), respectively. An alternative method forvector or random-access scanning a laser beam is to use two orthogonallymounted acousto-optic deflectors, (Iyer et al. (2002) Proc. 2nd AnnualJoint EMBS/BMES Conference, Houston, Tex., Oct. 23-26, 2002, 2101-2). Asdepicted in FIG. 3, the light energy is delivered to the user via minors74, scan lenses 76, and minor or beam splitters 78. Spectacle lens 80may be attached to the front of the headpiece to darken the user's eyesand/or add aesthetic effect to the headpiece.

Because system 60 uses LEDs for generating the light energy, the systemis extremely robust and can be battery powered and completely portable.The system is also produced at a much lower cost. For example, CreeInc., (Durham, N.C.) manufactures high-brightness edge-emitting LEDs forretinal-scanning display applications for Microvision Inc. (Bothell,Wash.), and Nichia Corporation (Tokyo, Japan) manufactures high-power UVLEDs that have a 100 mW source producing 380 nm light. Most all LEDs areextremely durable and have long lifetimes (greater than 50,000 hours).

The source size for LEDs is large enough to have more than a singleoptical fiber coupled to the emitter. To improve coupling efficiency,the fibers can have a larger core diameter than the singlemode opticalfibers depicted in FIGS. 1 and 2.

While system 60 may be lower in cost, presently available componentsrender it unable to produce images of highest resolution and brightnessas compared to the system 10 in FIGS. 1 and 2. The larger the size ofthe fiber optic source, the larger the imaged spot will be at theretinal neurons. Hence, LED-based system 60 may have a lower visualresolution and/or image brightness that is perceived.

Cameras 62 are shown mounted on the spectacle frame and are capable ofproducing still or continuous video images. Suitable embodiments of thecameras include “lipstick-style” CCD (obtainable from ELMO USA,Painview, N.Y.) or “sugar-cube” CMOS camera heads (obtainable from ICMedia Corp., San Jose, Calif.). One or more cameras may alternatively bemounted on the body of the user, as shown in FIGS. 1 and 2. Imageprocessor 64 receives the image signal from cameras 62 and modulates thelight energy produced by the LEDs in light energy generator 68. Imageprocessor 64 also controls the drive signals from can controller 66 sothat the X-Y scanner is synchronized with the light energy modulation.As with system 10 depicted in FIGS. 1 and 2, the spatially andintensity-modulated photostimulation generates two-dimensional imagesthat are perceived by the brain, thus mimicking a functional humanretina. Further, as with system 10 in FIGS. 1 and 2, system 60 may usefiber-scanning technology to produce the scanned image, in place ofmirror scanning system 72 shown in FIG. 3.

Compared to system 10, system 60 is less efficient in terms ofphotostimulation but is lower in cost and is more durable. Also, asnoted above, single photon LED neurostimulation may use larger coreoptical fibers, producing larger light spots (and consequent lowerresolution images) at the retina. Thus, optical fibers 70 may bemultimodal, as opposed to the singlemode fibers depicted in FIGS. 1 and2.

In the systems depicted in FIGS. 2 and 3, the optical and electroniccomponents may be carried in either a waist belt, backpack, vest withpockets, or otherwise clipped or strapped to the body. The portablesystems will be powered by batteries. An exemplary wearable computerthat may be used as image processor 64 is available from Xybernaut ofFairfax, Va.

In all embodiments of the invention, the image that is processed andscanned on the user's retina may come from a wide variety of sources,including video or still frame cameras that capture visible and/ornon-visible wavelength light. The sources may also includecomputer-generated signals, television signals, video tape, DVD, etc.(for example, block 41 in FIGS. 1 and 2). FIG. 3 further depicts anoptional connection 63 to image processor 64 for connecting analternative image source (in place of or in addition to cameras 62).

The most optically-efficient means for scanning an image to the retinais to convert the image source from a raster format to a vector graphicsformat, as shown in FIG. 4. Typically, video cameras 90 and electronicdisplay devices 92 use raster scanning format 94, where the scan coversa fixed area and pixels of the image are either turned on or off withgraded intensity and optionally color. Since photon energy is being usedto modulate neural activity in the retina, the most efficient use of thestimulating light is important. Otherwise, the eye can warm up due toexcessive optical radiation, possible damaging the melanin-containingretinal pigmented epithelium. Vector scanning 96 either drives a fiberscan system or mirror scan system to follow a path in X-Y space, similarto how an image is hand-drawn. As a component to a vision prosthesis,the image source will be reduced in detail by first enhancing edges ofall objects or segmented objects and then thresholding the color orgrayscale image into binary (On or Off) before converting the image to avector graphics format and possible rescaling (Vargas-Martin & Peli(2001) Soc. Information Display Tech. Digest 32:602, FIG. 1). Software98 is commercial available for making the conversion from raster tovector graphics format (e.g., Algolab Photo Vector, Algolab, Ontario,Canada).

Fiber scanner 28 may be connected to wearable headset 12 in acantilevered configuration and, as noted above, may be driven by one ormore piezoelectric actuators in either a resonant raster scan or a X-Yaddressable vector scan. A laser-diode-based, wearable fiber-scanneddisplay has been developed for low vision applications (Seibel et al.(2002) Soc. Information Display Tech. Digest 33(1):338-341). Alternativeretinal scanning display using resonant minor scanning has beenvalidated as a low vision aid (Kleweno et al. (2001) J. Rehab. Res. Dev.38(4):431-42). A femtosecond-pulsed NIR fiber-scanned system that iswearable by an animal has been developed (Helmchen et al. (2001) Neuron31:903-12). Lens 32 or lens system may be suitably adjusted for theprescription or eye refraction of the user.

When using lower-power UV or visible laser diodes (that are directlymodulated as discussed above), the output of two or more laser diodesmay be combined using a polarization cube beam splitter or combiner. Theoutput of a laser diode may be communicated via apolarization-preserving singlemode optical fiber through a collimatinglens into the cube beam splitter or combiner. Similarly, the output ofanother laser diode may be communicated via a polarization-preservingsinglemode optical fiber through another collimating lens into the cubebeam splitter or combiner. The combined output of the laser diodes isthen directed from the cube beam splitter or combiner through acollimating lens into a singlemode optical fiber (see products of OZOptics Corp., Carp, Ontario). The combined beams from the laser diodesmay have mixed parallel and perpendicular linear polarizations that maydouble the optical power output of the system. Using two or more laserdiodes, in this respect, may also provide redundancy in case of failureof a laser diode. In the above described system, a dichroic beamsplitter may be used in place of the cube beam splitter or combiner. Afiberoptic combiner may be used as well (see Canadian Instrumentation &Research Ltd., Burlington, Ontario; SIFAM Fibre Optics Ltd., Devon, UK).

Representative examples of apparatus that may be adapted for use in thepractice of the present invention to scan beams of light energy onto auser's retina, and thereby modulate neural activity in the retina, aredisclosed in U.S. Pat. Nos. 5,355,181; 5,467,104; 5,596,339; 5,727,098;and 6,046,720, the disclosures of which are incorporated herein byreference. It is also contemplated within the scope of the presentinvention to use the visual prosthesis to stimulate neurons other thanthose in the retina. For instance, the imaging system of the inventionmay produce electric signals that modulate neural activity relating toother senses, such as hearing, smell, taste, or touch, thus producing anartificially-formed sense for the user of the prosthesis.

To produce the smallest optical spot size—and thus improve imageresolution—in the user's retina, the present invention furthercontemplates fitting the user with a contact lens or an implantedintraocular lens (IOL) that is customized for this application. FIG. 5shows a representation of a mammalian eye 100 including the cornea 102,the iris 104 (dilated), a crystalline lens 106, the retina 108, and animplanted IOL 110. The natural pupil diameter, when less than about 2.7mm, limits the numerical aperture (NA) or focusing ability of eye 100,while aberrations in the cornea 102 and crystalline lens 106 limit theNA (resulting in reduced resolution and contrast of the perceived image)when the pupil is larger than about 2.7 mm. A large NA benefits directstimulation of retinal neurons using the system and methods of theinvention by producing smaller spots of irradiation on retina 108. Meansto increase the NA of eye 100 include increasing the pupil diameter(e.g., dilation) and correcting for optical aberrations in eye 100 witheither a custom contact lens or IOL 110 (e.g., a custom GRadient INdexoptical lens to replace natural crystalline lens 106). Such customizedlenses can correct for higher order aberrations of the user's eyes atthe wavelengths of the pulsed light energy generator (e.g., the laser)and the degree of collimation of the optical beam(s), and also reducescatter and absorption. Anti-reflection coatings may be applied to thecustom lens surfaces to reduce unwanted surface reflections. In an eye100 whose retina 108 is completely unresponsive to light, it may bedesirable to completely remove iris 104 and crystalline lens 106 andreplace them with a custom scan lens system in order to increase the NAof the incoming light energy that is being focused directly on theneurons of retina 108, especially for the high spatial density ofretinal neurons near the foveal and macular regions. In addition,permanent changes to cornea 102 may include laser sculpting for optimalfocus of the wavelength and beam quality of the light energy used in thesystem and methods of the invention.

If UV light is used for the stimulation of the retinal neurons, then theuser will require a standard IOL replacement since the naturalcrystalline lens does not transmit efficiently in the UV. Standard IOLreplacements may also transmit infrared light efficiently. However, theymay not be optimized for the laser wavelengths used for multi-photonphotostimulation of retinal neurons so a custom IOL may be desirable.

Thus, in some embodiments of the invention crystalline lens 106 from oneor both eyes of a mammalian subject may be removed and replaced withartificial lens 110 that is better adapted to focus light energy ontoretina 108. The ability to design and make a custom contact lens forenhanced vision has been recently demonstrated (Guirao et al. (2001) J.Opt. Soc. Am. A. Opt. Image Sci. Vis. 18(5):1003-15, incorporated byreference herein). Accordingly, a customized contact lens or intraocularlens (IOL) may be designed to improve the efficiency of the opticalradiation delivered by the visual prosthesis. Since both maculardegeneration and cataracts are age-related ocular diseases, the IOLreplacement of the crystalline lens for cataracts may coincide with theimplementation of the visual prosthesis of the present invention.Customized lenses may also be configured to correct for higher-orderaberrations in the user's eyes. In this manner, the focal spot at theuser's retinal neurons may be optimized (see discussion of customizedcontact lenses in Guirao et al. (2001) J. Opt. Soc. Am. A. Opt. ImageSci. Vis. 18(5):1003-15). For example, IOL lens implants are beingdesigned with adaptive optics for user's to compensate for natural lensaberrations (Hogan et al. (2003) Biophotonics Intl. August 2003, p.34-7). This concept may be extended to IR for efficient photostimulation.

In age-related macular degeneration, the natural photoreceptors in themacular region of the eye either partially or fully fail to function.However, photoreceptors in the peripheral regions may continue tofunction properly. Accordingly, the higher-power photostimulationprovided by the visual prosthesis of the invention is directed to themacular region of the eye. Light energy directed to the periphery of themacula may originate with low-power visual laser light sources tostimulate the functional photoreceptors, while the higher power laser isfocused on the macular region for direct modulation of retinal neuralactivity. The visible lasers may include the combined beams of red,green, and blue wavelength ranges to provide full color to thefunctional photoreceptors, while one of these lasers or afemtosecond-pulsed NIR laser provides the laser power for stimulatingthe region of non-functioning photoreceptors. Thus, the neuralstimulators may be composed of natural or artificial elements.Particularly in the case of a user having a region of functionalphotoreceptors, the option of gaze tracking or eye-movement monitoringmay be desirable. In the simplest case, only when the user'sstraight-ahead gaze is aligned with the optical axis of the device willphotostimulation of the retinal neurons occur. Optical sensors orcameras are typically used for gaze tracking or more simplygaze-alignment monitoring, such as using the reflected red-pupil signalthat occurs when the gaze is roughly aligned with a white light source,often seen in flash photographs.

The natural eye of a human has a numerical aperture (NA) ofapproximately 0.2 or less for a pupil of 5 to 6 mm in diameter. In orderto improve resolution of scanned images, the retinal neurons may bestimulated by light energy directed through an implanted intraocularlens of higher NA, as described above. In that regard, as noted above,it is contemplated to replace the entire optics of the eye with areplacement that functions as a high NA objective for the light energystimulation of the retinal neurons. The eye replacement may be designedto look like a natural iris and sclera, to be aesthetically pleasing forpersons interacting with the blind person. The high NA replacement inthis regard may be on the order of 0.7, i.e., at least three to fourtimes the NA of the natural eye. Artificially raising the NA of the eyemay significantly increase the efficiency of multiphoton excitation oroptoporation at the retina because the probability of two-photonexcitation is proportional to the fourth power of NA (Denk et al. (1990)Science 248:73-6.)

As an alternative to scanning light energy onto the retina, ahigh-intensity beam of light may be expanded to fill a spatial lightmodulator, such as liquid crystal on silicon chip (LCOS) or aMicroelectromechanical system such as the Digital Multimirror Device(DMD, Texas Instruments Inc.). Instead of having to retrace the entirescan every frame, the pixel-array device modulates the intensity of eachpixel in the display at the same time, which is updated every frame. Thespatial light modulation creating an image on the surface of thepixel-array device is re-imaged onto the retina to form the imagestimulus. In this alternative method, the optical beam is not scannedand illumination from several high-brightness LED's may providesufficient optical power for modulating neural activity. Since allpixels in each image are presented simultaneously using the DMD or LCOSpixel array chips, the preferred method of illumination using the LEDlight source(s) will be in a pulsed mode. Pulsing LEDs can provide muchhigher peak powers than continuous operation of LEDs.

As shown in FIG. 6, an array of light sources 120 (e.g., atwo-dimensional array of superbright LEDs, reviewed in Boas (2003)Biophotonics Intl., p. 27-28) that are individually-addressable anddirectly-modulateable in light output, which can be re-imaged andminified directly onto the retinal neurons of the eye, may simplyreplace the planar spatial light modulators (LCOS and DMD chips).Because each light source is individually addressable, complex patternscan be projected into eye 100. Projection lens 122 (e.g., with ¼magnifying power) minifies an image of the array of light sources 120 inorder to project a high-resolution pattern of irradiation on retina 108.The advantage of having an array of light sources is that each lightsource can stimulate the same region of the retina for the duration ofthe frame, which is usually 33 ms to 10 ms for video display frame ratesof 30 to 60 Hz. For this reason, scanning and sweeping the light ontothe retinal neurons across the entire field of view of the image is notrequired. However, image resolution (pixel number) is limited by thenumber of light sources in the array. To increase the number ofphoto-excited pixels, the array of light sources or the array of opticalbeams from a microlens array can be scanned.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

Example 1

This Example describes the optoporation of ganglion cells in embryonicchicken retinas using near-infrared irradiation.

Extraction of Chicken Embryos: Embryonic chicken retinal explants wereused for all experiments. Eggs were kept in an incubator untilimmediately prior to use. The contents of each egg were transferred to aPetri dish where the embryo was separated from the rest of the eggcontents using sterile forceps. The embryo was then transferred to aseparate dish containing Hank's Buffered Saline Solution (HBSS). Duringthe separation and transfer stages, the embryo was shoveled about,rather than pinched with the forceps. This process was repeated untilthe proper number of embryos had been collected.

Once all of the embryos had been extracted, they were viewed under adissection scope to determine their stage using a staging manual. Thestage of the embryos varied from experiment to experiment but werealways in the range of stage 29 to 41.

Dissection of Embryonic Chicken Retinas: Direct contact with the eye wasminimized during dissection in order to avoid accidentally damaging theeye. Once the eyes were separated from the rest of the embryo, they weretransferred to a new Petri dish containing HBSS. Once the eyes weretransferred to a new dish, extraction of the retinas followed.Dissection of the eye itself began with the removal of the cornea andthe lens. The eye was then splayed out to ease separation of the retinafrom the retinal pigmented epithilium (RPE). The act of splaying the eyecaused parts of the retina to detach from the RPE. These initial tearsprovided space to hold the eye immobile while only pinching the RPE andouter layers. While the eye was held immobile, the retina was gentlyteased away from the RPE by sliding a set of forceps between the retinaand the RPE (not by pinching the retina and pulling it). The retinaswere then transferred to a third dish containing HBSS. The retinas werequartered using forceps. Each quarter was then transferred to acoverslip chamber. Retinas were stored at 37° C. for a maximum of 5hours in 0.5 ml of HBSS.

Near-Infrared Irradiation of Retinas: All experiments were performed atthe University of Washington's Keck Imaging Center. Cell membranes weretargeted using a confocal laser scanning microscope (TCS SP/MP, LeicaMicrosystems AG, Wetzlar, Germany). The Leica TCS is an invertedmicroscope. Two-photon 2P optoporation experiments were performed usinga Spectra-Physics Tsunami Ti:Sapphire laser. The laser has a tuningrange of 720 nm to 850 nm, a maximum power at the sample of 120 mW, apulse width of 1.3 ps, and a repetition rate of 82 MHz. 2P experimentswere performed using near-infrared (NIR) wavelengths in the range of720-810 nm.

Immediately before the beginning of each experiment, green-fluorescentprotein (GFP) plasmid DNA (Qiagen, Wetzlar, Germany) was added to theHBSS to a final concentration of 25 micrograms/ml.

Prior to irradiation with the 2P laser, images of the cells wereobtained using Differential Interference Contrast (DIC) imaging. DICimages were collected using reflected light at 488 nm from an Argonlaser. The DIC images were used to bring the edge of cell membranes intofocus. Once a focused DIC image was obtained, an advanced time lapseseries was started. This series consisted of a DIC image taken with the488 nm laser, then a bleach experiment with the 2P laser (irradiationtime ˜500 ms), then another DIC image with the 488 nm laser. The bleachexperiment on the Leica TCS system consists of an interactive dialoguebox where the user can pick a specific point for the Leica Software topark the beam for a specified duration in time. Points in the middle ofthe retinal explant were targeted.

Following irradiation, the retinas were incubated rolling for 18 hoursat 37° C. in tissue culture media (20 ml 5×DMEM F-12, 2 ml 30% Glucose,1.5 ml 7.5% NaHCO₃, 0.5 ml 1 M HEPES, 65 ml sterile water, 10 ml HormoneSupplement, 1 ml FBS, and 1 ml of Penn/Strep). Retinas where then fixedusing 4% paraformaldehyde for 1 hour at room temperature. The tissue wasthen rinsed twice for 5 minutes with Phosphate Buffered Saline (PBS).Following the second rinsing, the tissue was mounted on microscopeslides using either Fluoromount or Glycerol. The slides were then storedat 4° C. until checked for fluorescence.

Fluorescence Microscopy: Tissue fluorescence was determined using afluorescence microscope. Images were collected on a Macintosh computerusing Spot and edited using Adobe Photoshop. The transfected cells weredetermined to be ganglion cells by the cell morphology. Based onfluorescence microscopy, the overall transfection efficiency wasdetermined to be 42%.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A vision prosthesis system, comprising a light energy generatoroperably connected to a wearable head piece comprising a device fordirecting light energy produced by the light energy generator onto amammalian retina, wherein the light energy generator is tuned to emitnon-visible light energy of sufficient power to directly modulate neuralactivity in the retina in neurons other than rods and cones, and whereinthe power level of light energy directed onto the mammalian retina isgreater than about 1 mW, and further comprising means adapted to directthe non-visible light energy directly onto the mammalian retina.
 2. Thevision prosthesis system of claim 1, wherein the light energy generatoris tuned to emit infrared light energy.
 3. The system of claim 1,wherein the light energy generator is tuned to emit ultraviolet lightenergy.
 4. The system of claim 1, wherein the means for directing thenon-visible light energy directly onto the mammalian retina comprises awearable head piece, a fiber optic cable for transmitting light energyfrom the light energy generator to the wearable headpiece, and a lensfor focusing light from the fiber optic cable onto the mammalian retina.5. The system of claim 1, wherein the light energy generator is selectedfrom the group consisting of a laser, a laser diode, a mercury arc lamp,and a light-emitting diode.
 6. The system of claim 1, wherein the devicefor directing light energy onto the mammalian retina is a scanner.